Allosteric Properties, Substrate Specificity, and Subsite Affinities of Honeybee α-Glucosidase I

抄録

The substrate specificity of honeybee α-glucosidase I, a monomeric enzyme, was kinetically investigated. Unusual kinetic features were observed in the cleavage reactions of sucrose, maltose, p-nitrophenyl α-glucoside, phenyl α-glucoside, turanose, and maltodextrin (DP=13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl α-glucoside, and phenol from phenyl α-glucoside were accelerated, and so the Lineweaver-Burk plots were convex, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, 0.50, and 0.67 for sucrose, maltose, p-nitrophenyl p-glucoside, and phenyl α-glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Burk plots were concave and the Hill coefficients were 1.2 and 1.5 for turanose and maltodextrin, respectively. These unique properties cannot be interpreted by the reaction mechanism that Huber and Thompson proposed: (1973) Biochemistry 12, 4011-4020. The rate parameters for the hydrolysis of sucrose, maltose, p-nitrophenyl α-glucoside and phenyl α-glucoside were estimated by extrapolat-ing the linear part of the Lineweaver-Burk plots at low substrate concentrations. The ratios of the V_max values for maltose, sucrose, kojibiose, p-nitrophenyl α-glucoside, phenyl α-glucoside, phenyl α-maltoside, and malto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose, were estimated to be 100: 95 : 81 : 126: 85 : 86 : 113: 117: 111 : 104: 95 : 95, and the K_m values for these substrates were 0.85, 4.2, 25, 0.31, 0.60, 0.41, 0.62, 2.0, 8.0, 15, 30, and 33mM, respectively. Based on the rate parameters for maltooligosaccha-rides, the subsite affinities (A_is) in the active site of the enzyme were evaluated. Subsites 1, 2, and 3 having positive A1 values (A₁, A₂, and A₃ : 1.34, 5.37, and 0.269 kcal/mol, respective-ly) were considered to be effective for the binding of substrate to the active site.